This invention relates to the construction and usage of synthetic genes for genetic engineering and gene therapy.
Recombination at the genetic level is important for generating diversity and adaptive change within genomes of virtually all organisms. Recombinant DNA technology is based upon simple xe2x80x98cut-and-pastexe2x80x99 methods for manipulating nucleic acid molecules in vitro. The pieces of genetic material, or DNA are first digested with a restriction endonuclease enzyme which recognizes specific sequences within the DNA. After preparation of two or more pieces of DNA, the ends of the DNA are further manipulated, if necessary, to make them compatible for ligation or joining together. DNA ligase, together with adenosine triphosphate (ATP) is added to the genes, ligating them back together. The genetic assembly containing an origin of DNA replication and a selectable gene is then inserted into a living cell, is grown up, and is positively selected to yield a pure culture capable of providing high yields of individual recombinant DNA molecules, or their products such as RNA or protein.
Significant improvements have been made to this technology over the last two and a half decades. Numerous enzymes, end-linkers and adapter molecules have been made commercially available, which facilitate in the construction of recombinant DNA molecules. By using two restriction enzymes with different single-stranded termini or blunt ends, it is possible to directionally assemble genes (forced cloning). This reduces the amount of screening required to determine orientation. Procedures have been automated for synthesis of single-stranded gene fragments up to 200 or more nucleotides in length by means of phosphoramidite chemistry, and the instrumentation is readily available through Applied Biosystems, Inc., Foster City, Calif. Such single-stranded fragments can be joined by annealing overlapping complimentary phosphorylated strands, and by enzymatically filling in the ends with DNA polymerase and DNA precursors In this way, multiple, overlapping, single-stranded fragments can be assembled into a larger, double-stranded superstructure. Whole genes have been synthesized by similar methods. However, it becomes increasingly difficult to use synthetic DNA strands when making genes larger than approximately one kilobase. Using gene amplification methods (e.g. polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,195), together with synthetic oligonucleotides, it is possible to make biologically active, synthetic retro-vectors that are capable of RNA transcription, reverse-transcription, viral packaging, and integration into genomic DNA (see for example, Hodgson, WO94/20608). Hodgson, supra, also disclosed methods for cloning of transcriptional promoters into such a vector using traditional recombinant DNA technology.
Modified restriction enzyme sites, linkers, and adapters can change the primary or secondary structure of complex nucleic acid sequences thereby altering or obliterating a desired biological activity. For example, small mutations can drastically modify transcriptional promoters or change the reading frame of coding DNA. A logical goal of vectorology is to make exact constructs, without need of fortuitous restriction sites, adapters, or linkers.
Restriction endonucleases can be grouped based on similar characteristics In general there are three major types or classes: I, II (including IIS) and III. Class I enzymes cuts at a somewhat random site from the enzyme recognition sites (see Old and Primrose, 1994. Principles of Gene Manipulation. Blackwell Sciences, Inc., Cambridge, Mass., p.24). Most enzymes used in molecular biology are type II enzymes. These enzymes recognize a particular target sequence (i.e., restriction endonuclease recognition site) and break the polynucleotide chains within or near to the recognition site. The type II recognition sequences are continuous or interrupted. Class IIS enzymes (i.e., type IIS enzymes) have asymmetric recognition sequences. Cleavage occurs at a distance from the recognition site.
These enzymes have been reviewed by Szybalski et al. Gene 100:13-26, 1991. Class III restriction enzymes are rare and are not commonly used in molecular biology.
U.S. Pat. No. 4,293,652 employed a linker with a class IIS enzyme recognition sequence to permit synthesized DNA to be inserted into a vector without disturbing a recognition sequence. Brousseau et al. (Gene 17:279-289, 1982) and Urdea et al. (Proc. Natl. Acad. Sci. USA 80:7461-7465, 1983) disclose the use of class IIS enzymes for the production of vectors to produce recombinant insulin and epidermal growth factor respectively. Mandecki et al. described a method for making synthetic genes by cloning small oligonucleotides using a vector (Gene 68:101-107, 1988). Expansion of a population of oligonucleotides required synthesis, cloning excision and fragment purification. The oligonucleotides were used to create a complete plasmid.
Lebedenko et al. (Nucl. Acids Res. 19(24):6757-6761) illustrated the class IIS enzymes and PCR for precisely joining 3 nucleic acid molecules for convention sub-cloning using BamHI. Tomic et al. (Nucleic Acids Res., 18:1656, 1990), reported a method for site-directed mutagenesis using the polymerase chain reaction and class IIS enzymes to join two nucleic acid molecules. Two overlapping PCR primers were used where the primers included class IIS recognition sites. The primers included a region of complementarity to the template DNA and include one to a few site-directed mutations. Stemmer (U.S. Pat. No. 5,514,568) employed overlapping primers with class IIS enzymes to amplify a plasmid and to introduce specific mutations into DNA leaving all other positions unaltered.
There remains a need for the ordering and assembly of complex genes to overcome the problems associated with sequential sub-cloning such as multiple purification steps, the potential for sample loss, and the like. Moreover there is a need for eliminating the use of prokaryotic hosts and for minimizing or avoiding the risks associated with bacterial contamination resulting from the use of bacteria as intermediaries in the cloning process. Further, there remains a need for efficient methods to assemble large nucleic acid molecules or many-fragmented nucleic acid assemblies with precision.